BIOSYNTHESIS OF 3,4-DIDEHYDRORETINOL FROM RETINOL BY HUMAN SKIN KERATINOCYTES IN CULTURE

The uptake and metabolism of radiolabelled retinol was studied in cult ivated human skin cells. Normal epidermal keratinocytes in primary cul ture were able to incorporate unbound [11, 12-H-3]all-trans-retinol fr om the growth medium and transform it into 3,4-didehydroretinol (dehyd roretinol) in a dose- and time-dependent manner. A total of 23% of the radioactive label became cell-associated during a 48-h incubation per iod when added at 7 nM to differentiated keratinocytes submerged in se rum-containing, high-calcium (1.56 mM) culture medium. At that time po int, 25-30% of cell-bound radioactive retinol had been converted into dehydroretinol, with no labelled retinal, dehydroretinal, retinoic aci d or dehydroretinoic acid being detected in cells or medium. Thus dehy droretinol, which occurs physiologically in mammalian skin tissue in v ivo, was identified as the predominant neutral retinol metabolite in c ultured keratinocytes using h.p.l.c. and anhydro-derivatization proced ures. At least 94% of the product, along with its precursor, was prese nt in the cells in esterified form, with no traces of the compound bei ng secreted into the cell environment. The rate of formation of dehydr oretinol from its precursor was significantly lower in keratinocytes g rown in serum-free, low-calcium (0.09 mM) culture medium. and in mediu m pre-incubated with excess unlabelled substrate. Furthermore, the app lication of 13-cis-retinoic acid (isotretinoin), a therapeutic retinoi d drug known to markedly reduce dehydroretinol levels in human skin, b locked the biosynthesis of this metabolite in cultured keratinocytes. The 3,4-dehydrogenation pathway observed in this study could not be sh own to operate to any significant extent in cultures of human epiderma l melanocytes or dermal fibroblasts, supporting the hypothesis that ke ratinocytes represent the principal cell type involved in dehydroretin ol formation from retinol in human skin.